Transducing phages of Actinomycetales

ABSTRACT

The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including  Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis,  and  Saccharopolyspora erythraea.  The transducing phages can be specialized transducing phages or generalized transducing phages.

This patent application claims the benefit of U.S. provisional patentapplication No. 60/126,391, filed Mar. 26, 1999.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.1021RR093161, awarded by the National Science Foundation. The Governmentmay have certain rights in this invention.

BACKGROUND OF THE INVENTION

It would be difficult to overestimate the contribution generalizedtransduction has made to the study of prokaryote biology since thediscovery of phage P22 in Salmonella in the early 1950s. The use ofgeneralized transducing phages for strain construction, fine structuremapping, and genetic manipulation have played major roles in the geneticanalysis of Salmonella and E. coli. One of the most importantapplications of generalized transduction has been to facilitate thecloning of genes identified by transposon generated mutations. The useof generalized transduction in combination with transposon mutagenesisto clone genes involved in morphogenesis has been invaluable in thestudy of sporulation in Bacillus subtilis.

Streptomyces are Gram-positive soil bacteria of special interest for tworeasons. First, their mycelial growth mode and sporulation cycle areamong the most dramatic examples of prokaryotic morphologicaldifferentiation. They grow vegetatively as multicellular, multinucleoid,branching hyphae that penetrate and solubilize organic material in thesoil forming a mycelial mass. In response to environmental signals (aprocess that requires cell—cell communication mediated by diffusiblesubstances), they initiate a cycle of differentiation that begins withthe production of aerial hyphae that septate into uninucloidcompartments that give rise to spores. Second, during the initiation ofmorphological development they produce a large number of secondarymetabolites, including most of the natural product antibiotics used inhuman and animal health care. Because of its unique biology,Streptomyces offers special advantages for the study of howmorphogenesis is initiated. The question of how cells withinmulticellular organisms sense changes in their environment andcommunicate that information to each other is of fundamental importanceto the study of developmental biology. In spite of their interestingbiology and commercial importance, relatively little is known about thegene expression pathways that regulate morphological development orantibiotic biosynthesis.

A major limitation in the study of Streptomyces is that the typicalgenetic approaches for recovering genes identified by chemically inducedmutations have been difficult to implement in Streptomyces. Becauserelatively few genetic markers exist in Streptomyces, fine structuremapping is not possible. Cloning by complementation is slow and tedious.Transformation of plasmid libraries constructed in either E. coli orStreptomyces is extremely inefficient and the libraries are oftenincomplete. Transposition systems have been developed in Streptomycesbut they have not proved to be effective for insertional mutagenesis.This is in part due to the use of temperature sensitive plasmid vectorsas transposon delivery systems. Plasmid curing is not effective andexposure to high temperatures is mutagenic in itself. This has resultedin a high background of mutations not caused by transposition. Thus, ithas not been possible to determine whether a mutant phenotype was causedby transposon insertion into a gene of interest until the candidate genewas cloned, thereby permitting complementation analysis and directeddisruption studies. This is not only time consuming and laborious, it isoften a futile exercise because of the high background of extraneousmutations.

It has long been recognized that an efficient system for generalizedtransduction is needed to make transposon mutagenesis an effectivegenetic tool in Streptomyces. However, generalized transducing phageshave not been characterized in species that can serve as genetic modelsystems. Attempts by many workers over the years to isolate generalizedtransducing phages for Streptomyces coelicolor have been uniformlyunsuccessful, as have been attempts to transduce markers by the mostextensively studied lytic actinomycete phages fC31, VP5, and R4.Generalized transduction has been demonstrated in Streptomycesvenezuelae. This involved transduction of several markers includinggenes for cholemphenicol production. This was thought, however, to be ananomaly and somehow specific to Streptomyces venezuelae since theapproaches used to identify transducing phages for Streptomycesvenezuelae did not work for Streptomyces coelicolor.

Subsequent to the publication of much of the work describing theseintraspecific generalized transducing phages of Streptomyces venezuelaeand Streptomyces olivaceus, a report was authored by one of theinvestigators that had taken part in many of the studies. In this reporttitled “Generalized Transduction in Streptomyces Species,” (Stuttard,In: Genetics and Molecular Biology of Industrial Microorganisms,Hershberger, et al., (eds.), pp. 157-162, ASM, Washington, D.C. (1989))he reported “a possibly significant lack of success with Streptomycescoelicolor and Streptomyces lividans.” The author hypothesized “thatsome essential host function(s), possibly expressed in few potentialhost strains, may be required for lytic growth of” generalizedtransducing particles. If such host functions are required, thengeneralized transducing phages will not be isolated that transduce thosestrains lacking the essential host functions. The author concludes that“generalized transducing phages for Streptomyces coelicolor andStreptomyces lividans remain as elusive as ever.”

In the recent past there has been a significant increase in theidentification of antibiotic resistant microbes. However, theidentification of new antibiotics has not kept pace with the occurrenceof antibiotic resistant microbes. Accordingly, there has been asignificant increase in human and animal morbidity and mortality due toinfectious diseases. Thus, there is a need for new antibiotics. Asmentioned above, Streptomyces, and other microbes, produce secondarymetabolites. Many of these secondary metabolites are natural productantibiotics used in human and animal health care. It has recently becomepossible to use recombinant genetic techniques to modify the metabolicpathways of microbes to result in the synthesis of new natural productantibiotics, often referred to as new natural products or non-naturalproducts, having new activities. A limitation to this is, for instance,the need for appropriate vectors to carry large DNA fragments, and theability to efficiently move DNA into appropriate hosts (see, forinstance, Cane, D. E. et al., (1998) Science, 282, 63-68). Thus, thereis a need and significant advantage to developing genetic techniques ofmicrobes that synthesize natural product antibiotics.

SUMMARY OF THE INVENTION

The present invention is directed to a method of isolating a transducingphage, preferably, a generalized transducing phage. The method includescombining a sample containing a transducing phage with a microbe forminga first phage-microbe mixture, and incubating the first phage-microbemixture at a temperature of less than 28° C. to form a first plaquecomprising a generalized transducing phage. The invention includes aphage isolated using this method.

Another aspect of the invention is a method of isolating a transducingphage, preferably, a generalized transducing phage, involving phage DNA.The method includes combining a sample containing generalizedtransducing phage DNA with a microbe forming a phage DNA-microbe mixtureand incubating the phage DNA-microbe mixture at a temperature of lessthan 28° C. to form a first plaque comprising a transducing phage.

Another method of the invention is a method of transferring at least onenucleic acid fragment from a donor microbe to a recipient microbe. Themethod includes providing an isolated transducing particle comprising anucleic acid fragment from a donor microbe, combining the transducingparticle with a recipient microbe to result in a transducingparticle-recipient microbe mixture, and incubating the transducingparticle-recipient microbe mixture at a temperature of less than 28° C.to form a transduced recipient microbe comprising a nucleic acidfragment from the donor microbe. This method can also be used to producea secondary metabolite from a microbe. When a secondary metabolite is tobe produced, the method further includes providing conditions effectivefor the recipient microbe to produce a secondary metabolite. Theinvention also includes a microbe prepared by this method, and asecondary metabolite produced by this method.

The invention is also directed at an isolated generalized transducingphage that can transfer at least one nucleic acid fragment from a donormicrobe to a recipient microbe, wherein the frequency of transduction isat least about 10⁻⁷, and wherein the transduction of the recipientmicrobe occurs at less than 28° C.

A “phage” is able to inject a nucleic acid fragment into a host microbe.A type of phage is a “transducing phage.” When a transducing phageinfects a host microbe and replicates, two types of particles canresult. One type of particle produced during the replication process isa “phage particle.” As used herein, a phage particle contains a phagenucleic acid fragment and can infect another microbe and replicate, andcan therefore be used as a transducing phage. The second type ofparticle is a “transducing particle.” As used herein, a transducingparticle contains at least one nucleic acid fragment derived from thehost microbe. This distinction is important with respect to thediscussion of superinfection killing herein. Thus, as used herein, theterm phage is used generically to encompass phage that contain a phagenucleic acid fragment (i.e., a phage particle) or at least one nucleicacid fragment derived from a host microbe (i.e., a transducingparticle).

Transducing particles retain the ability to inject a nucleic acidfragment into a microbe. A microbe that is the recipient of a hostmicrobe nucleic acid fragment from a transducing particle is said to be“transduced,” and is referred to herein as a “transductant.”

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Phage susceptibility to UV irradiation. The log of the phagetiter is plotted versus time of exposure to UV irradiation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to isolated transducing phages andmethods of isolating transducing phages. The present invention is alsodirected to methods of using phages including, for instance,transferring at least one nucleic acid fragment from a donor microbe toa recipient microbe, and optionally producing a secondary metabolitefrom a microbe. The transducing phages can be specialized transducingphages or generalized transducing phages. Preferably, they aregeneralized transducing phages.

A phage can include a phage nucleic acid fragment (i.e., a nucleic acidfragment containing at least a portion of a phage genome) wrapped in aprotein coat. In nature, phages are not capable of growth outsidemicrobial cells. A phage adsorbs to a microbial cell via the proteins inthe coat and injects the nucleic acid fragment into the microbial cell.The phage nucleic acid fragment is replicated, transcribed, and thetranscipts are used to produce protein for the production of new phageparticles, i.e., more phage. Transducing phages are phages capable ofgenerating two types of particles. One type of particle, a transducingparticle, contains a nucleic acid fragment other than a phage nucleicacid fragment, e.g., a nucleic acid fragment from a host microbe. Thesecond type of particle, a phage particle, contains only a phage nucleicacid fragment (i.e., it does not include a nucleic acid fragment fromthe host microbe).

When the transducing phage is a generalized transducing phage, thenucleic acid fragment present in a transducing particle can originatefrom different areas of the genomic DNA present in the donor, or canoriginate from a plasmid present in the donor. When the transducingphage is a specialized transducing phage, the nucleic acid fragmentpresent in a transducing particle typically originates from one specificarea of the genomic DNA present in the donor.

A transducing particle can be produced naturally, i.e., it is the resultof the infection and subsequent lysis of a microbe infected with atransducing phage. A transducing particle can also be produced usingartificial methods, including, for instance, in vitro packaging offragmented genomic DNA. As used herein, “isolated” phage, phageparticle, or transducing particle refers to a phage separated from itsnatural environment. Preferably, an “isolated” phage, phage particle, ortransducing particle is a phage, phage particle, or transducing particlethat is separated from microbes and other phage, as opposed toessentially free from agar, cellular debris, and other impurities.

The phage of the present invention, preferably a generalized transducingphage, can transfer at least one nucleic acid fragment from a donormicrobe to a recipient microbe. A recipient microbe that has received atleast one nucleic acid fragment from a transducing particle is referredto as transduced. A “nucleic acid fragment” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxynucleotides, and includes both double- and single-stranded DNA(both genomic and plasmid) and both double- and single-stranded RNA. Apolynucleotide fragment may include both coding and non-coding regionsthat can be obtained directly from a natural source (e.g., a microbe),or can be prepared with the aid of recombinant or synthetic techniques.

Significantly and preferably, the phage of the present inventiontransduce at a temperature of less that 28° C. More preferably,transduction occurs at, in increasing order of preference, less thanabout 28° C., less than about 25° C., less than about 23° C., and lessthan about 21° C. It is expected that the lower limit of the temperatureat which transduction occurs is about 16° C. The low temperature isadvantageous because it allows a significant increase in the frequencyof transduction. The ability of the phage to cause transduction,preferably generalized transduction, at increased frequencies at atemperature of less that 28° C. was unexpected. Without intending to belimiting, it is believed that the decreased temperature of transductioncauses decreased superinfection, particularly superinfection killing, ofa transduced recipient. Other methods to decrease superinfection,particularly superinfection killing, are described herein.

Typically, the phage of the present invention transduce a recipientmicrobe at a frequency of transduction of at least about 10⁻⁷ (i.e., onetransduced recipient per 10⁷ phage). “Frequency of transduction” refersto the number of transduced recipients (i.e., transductants) per phageparticle after exposing a recipient strain to phage. Preferably,transduction occurs at, in increasing order of preference, at leastabout 10⁻⁶, at least about 10⁻⁵, and at least about 10⁻⁴. It isestimated that as high as about 10⁻³ can be achieved.

Preferably, the donor and recipient microbes are members of differentfamilies, more preferably, members of different genera, even morepreferably, members of different species, and most preferably, membersof the same species. This is referred to in the art as having a broadhost range.

Preferably, the Families are of the Order Actinomycetales. Preferably,the Families include Mycobacteriaceae, Actinomycetaceae,Streptomycetaceae, and Actinoplanaceae, more preferably,Streptomycetaceae. Preferably, the microbe is a spore, a mycelialfragment, a germling, a protoplast, or mixtures thereof. ManyActinomycetales naturally grow as a filament of cells. A mycelialfragment is a portion of this filament. A germling is a spore that isbeginning germination as determined by the appearance of germ tubes on aspore. Preferably, members of the family Streptomycetaceae that can betransduced by the phage are Streptomyces and Saccharopolyspora. Examplesof members the genus Streptomyces include Streptomyces coelicolor,Streptomyces lividans, Streptomyces venezuelae, and Streptomycesavermitilis. An example of members of the genus Saccharopolysporaincludes Saccharopolyspora erythraea.

The phage of the present invention, preferably a generalized transducingphage, can be isolated by combining a sample containing a transducingphage with a microbe forming a first phage-microbe mixture andincubating the first phage-microbe mixture to form a first plaquecomprising a transducing phage. Preferably, the incubation temperatureis less than 28° C. Typically, the plaques formed by the phage of thepresent invention are clear or turbid. A plaque refers to an area,typically but not necessarily in a solid or semi-solid bacteriologicalmedium, containing phage and lysed microbes. Typically, a plaque willalso contain unlysed microbes that may or may not be infected with aphage nucleic acid fragment. The lysed microbes have been lysed byinfection of a phage nucleic acid fragment, subsequent replication ofthe phage within the microbe, and then release of the replicated phageinto the surrounding area by lysis of the microbe. Typically, a plaquecontaining predominantly lysed microbes appears to be clear (i.e., nomicrobes are visible), while a plaque containing unlysed microbesappears as turbid (i.e., microbes are visible). The invention is furtherdirected to a phage prepared by this method. Preferably, phage preparedby this method is an isolated phage.

The microbe that is used to isolate a phage of the present invention canbe chosen from different families as described herein. Preferably, themicrobe is an Actinomycetales.

Typically, a source of divalent cations is present during a portion ofthe incubation of the isolation process. Preferable divalent cationsinclude transition metals and main group metals, and more preferably,calcium and magnesium. The sample containing a transducing phage,preferably a generalized transducing phage, can be obtained from thelithosphere and hydrosphere, including, for instance, soil, water,organic material, decomposing organic material, or volcanic ash.Preferably, a transducing phage is obtained from a composition thatincludes soil or volcanic ash, more preferably, soil.

Optionally, the isolation method includes separating the phage from thelithosphere or hydrosphere prior to combining the sample containing ageneralized transducing phage with a microbe. For instance, separatingthe phage from the lithosphere can include combining a sample containingthe phage, preferably a soil sample, with a diluent, preferablyincluding water, to form a slurry and removing particles that areheavier than the phage. Particles heavier than the phage can be removedby, for instance, centrifuging the slurry. The sample can be furthermanipulated to remove microbes. Preferably, microbes are removed byfiltration. Alternatively, microbes can be removed by adding an organicsolvent, preferably chloroform, to the sample containing the phage.

The isolation method can include (and typically does include) separatingthe phage from the plaque to form isolated phage. For instance, thephage can be separated from the plaque by combining the first plaquewith a microbe to form a second phage-microbe mixture and incubating thesecond phage-microbe mixture, preferably at a temperature of less than28° C., to form a second plaque containing a transducing phage. Thisstep can be repeated as many time as is necessary, preferably aboutthree times, to form an isolated phage. This process is typicallyreferred to as plaque purification.

An alternative method of isolating a transducing phage includesisolating phage DNA from a sample containing a transducing phage. ThisDNA can be combined with a microbe to form a phage DNA-microbe mixtureand incubating the phage DNA-microbe mixture at a temperature of lesstan 28° C. to form a plaque comprising a transducing phage. Preferably,the microbe is an Actinomycetales, more preferably a Streptomycetaceae,most preferably a Streptomyces. Preferably, the microbe is a protoplastfor this method of isolation.

The present invention is also directed to a method of transferring atleast one nucleic acid fragment from a donor microbe to a recipientmicrobe. Preferably, the donor and recipient are Actinomycetales, morepreferably a Streptomycetaceae, most preferably a Streptomyces. A methodof such a transfer includes providing an isolated transducing particlecomprising a nucleic acid fragment from a donor microbe. The transducingparticle can be combined with a recipient microbe to result in atransducing particle-recipient microbe mixture, and the transducingparticle-recipient microbe mixture incubated, preferably at atemperature of less than 28° C., to form a transduced recipient microbe,where the transduced recipient microbe contains a nucleic acid fragmentfrom the donor microbe. The invention is further directed to a microbeprepared by this method.

The method of transferring at least one nucleic acid fragment canfurther include reducing superinfection, preferably superinfectionkilling, of the transduced recipient microbe. Superinfection of atransduced recipient refers to a recipient containing a nucleic acidfragment from a phage particle and a nucleic acid fragment from atransducing particle. The presence of phage DNA from the phage particlewill typically result in lysis of the recipient. This is referred to assuperinfection killing. It is advantageous to reduce superinfection,preferably superinfection killing, of a transduced recipient to increasethe frequency of transduction.

Superinfection can be reduced by treating the transducing particle(which is typically in a suspension containing phage particles) prior tocombining it with the recipient microbe. Preferably, the transducingparticle-phage particle mixture is treated by exposing it to ultravioletradiation. Without intending to be limiting, it is believed that theultraviolet radiation inactivates the particles present. Since there istypically many more phage particles relative to transducing particles,more phage particles are inactivated. In general, appropriate conditionsfor using ultraviolet radiation include the time of exposure, thedistance of the particles from the ultraviolet source, and the media theparticles are in. Such conditions vary but can be easily determined by aperson of skill in the art. Preferably, the wavelength is about 250 nmto about 270 nm, and more preferably about 250 nm to about 260 nm.Preferably, the intensity is about 1.9 mW/cm²/s to about 2 mW/cm²/s, andmore preferably it is 2 mW/cm²/s.

Superinfection can also be reduced by treating the transduced recipientmicrobe with a chelator. Chelators useful in the present inventioninclude citrate and ethylene glycerol-bis(β-aminoethyl etherN,N,N′,N′,-tetraacetic acid (EGTA)). Preferably, the chelator is asource of citrate, such as sodium citrate. Chelators are preferably usedat a concentration that inhibits the ability of a particle to adsorb toa microbe, but does not significantly negatively affect the viability ofthe microbe. This concentration typically varies depending on thechelator used, but can be easily determined by a person of skill in theart. Typical concentrations of citrate are from about 1 mM to about 50mM, preferably about 10 mM. Superinfection can also be reduced bycombining low temperature and a chelating agent, or low temperature andultraviolet radiation, or all three.

An isolated transducing particle that includes a nucleic acid fragmentfrom a donor microbe can be obtained by several methods. For instance,an isolated phage, preferably a transducing phage, can be combined witha donor microbe to form a phage-donor microbe mixture. This phage-donormicrobe mixture can be incubated, preferably at less than 28° C., toform transducing particles. Alternatively, a transducing particle can beproduced using artificial methods, for instance, in vitro packaging.Preferably, the isolated transducing particle is provided in asuspension of phage comprising isolated transducing particles. Ingeneral, the higher the concentration of transducing particles that arecombined with a recipient microbe, the higher the probability of forminga transduced recipient microbe that contains a nucleic acid fragmentfrom the donor microbe. Preferably, the concentration of the transducingparticles in the suspension of phage is, in increasing order ofpreference, at least about 1 in 10⁸ (1 transducing particle in 10⁸ phageparticles), at least about 1 in 10⁷, at least about 1 in 10⁶, at leastabout 1 in 10⁵, at least about 1 in 10⁴, and at least about 1 in 10³.

A nucleic acid fragment from a donor microbe can contain a non-codingregion, a coding region or a portion thereof, or a mixture thereof.Preferably, the nucleic acid fragment from a donor microbe includes atleast one coding region. A “coding region” is a linear form ofnucleotides that typically encodes a polypeptide, usually via mRNA, whenplaced under the control of appropriate regulatory sequences (e.g., apromoter). The boundaries of a coding region are generally determined bya translation start codon at its 5′ end and a translation stop codon atits 3′ end, or a transcriptional start site at the 5′ end and atranslational stop codon or a transcriptional stop site at the 3′ end.

A coding region may encode a polypeptide or a transcript (i.e., an RNAtranscript) that is involved in the synthesis of a metabolite, orpolypeptides that impart antibiotic resistance or catalyze the synthesisof an antibiotic (e.g., lincomycin, or rifampicin). A metaboliteincludes primary metabolites (i.e., the products or intermediates of aprimary metabolic pathway), and secondary metabolites (i.e., products orintermediates of a secondary metabolic pathway). As used herein,“metabolic pathway” includes primary metabolic pathways and secondarymetabolic pathways. A “polypeptide” as used herein refers to a polymerof amino acids and does not refer to a specific length of a polymer ofamino acids. Thus, for example, the terms peptide, oligopeptide,protein, structural protein (e.g., one of several polypeptides in amultimeric complex) and enzyme are included within the definition ofpolypeptide. A polypeptide can be involved in, e.g., the catalysis of aproduct or intermediate, or the transport or anchoring of a product orintermediate. A polypeptide can also be involved in, e.g., holding amultimeric complex together, or post-synthesis steps of a product, e.g.,transport of a product.

Using the methods of the present invention, a recipient microbe can betransduced so that it expresses polypeptides not produced by therecipient microbe prior to transduction. Alternatively, a recipient canbe transduced so that it expresses a polypeptide in different amounts(increased or decreased) than the microbe could prior to transduction.This method is expected to allow the construction of microbes that havealtered metabolic pathways. This is sometimes referred to in the art as“metabolic engineering.” For instance, the transduced recipient microbecan produce a metabolite, a secondary metabolite, or a polypeptide at adifferent level, either higher or lower, than is produced by therecipient microbe prior to transduction. A nonlimiting example of thisis the transduction of a recipient to produce increased levels ofacetyl-CoA. In this transduced recipient producing increased levels ofacetyl-CoA it is further expected that the amounts of products ofmetabolic pathways that use acetyl-CoA will be increased.

The present invention can be used to transfer at least one nucleic acidfragment containing a coding region that encodes a marker, including,for example, one that can complement a mutation present in a recipientor encodes an antibiotic. When a marker that complements a mutation istransferred to a recipient, preferably the transduction of the markeroccurs at a frequency that is greater than the normal mutation rate(i.e., reversion frequency) for a marker. For example, as shown in theExamples, the reversion frequency of the arg mutation in a recipientstrain is <1 in 10⁺¹⁰, and the transduction frequency of a functionalarg marker is greater than the reversion frequency.

Another aspect of the present invention is directed to a method ofproducing a product or an intermediate of a metabolic pathway from amicrobe. Preferably, the method produces a secondary metabolite.Preferably, the donor and recipient are Actinomycetales, more preferablya Streptomycetaceae, most preferably a Streptomyces.

The method of producing a product or an intermediate of a metabolicpathway, preferably a secondary metabolite, from a microbe is similar tothe method of transferring at least one nucleic acid fragment from adonor microbe to a recipient microbe, as described herein. Whenproducing a secondary metabolite, the method can include providingconditions effective for the recipient microbe to produce the secondarymetabolite. The secondary metabolite produced by the transducedrecipient can be produced by the donor. The secondary metaboliteproduced by the transduced recipient microbe can be produced by therecipient microbe prior to transduction. Preferably, if the secondarymetabolite is the same as one produced by the donor or recipient microbeprior to transduction, the transduced recipient produces a secondarymetabolite at a higher level than is produced by the donor microbe orthe recipient microbe prior to transduction.

Alternatively and significantly, it is anticipated that the methods ofthe present invention will allow for the production of secondarymetabolites that are not produced by the recipient microbe prior totransduction or by the donor microbe, i.e., new secondary metabolites.New secondary metabolites are often referred to in the art as newnatural products, or non-natural products. The concept of the productionof secondary metabolites that are not produced by the recipient microbeprior to transduction or by the donor microbe is typically referred toin the art as combinatorial biosynthesis.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES

These examples detail the isolation of the first generalized transducingphages for Streptomyces coelicolor, the most genetically wellcharacterized strain of this important bacterial genus. Phages rangingin size from approximately 25 kb to more than 60 kb were shown totransduce a number of markers at frequencies from 10⁻⁵ to 10⁻⁸.Transduction is apparently general since markers were transduced fromlocations around the entire chromosome. Co-transduction of severalmarkers predicts linkage that is in good agreement with data obtainedfrom genetic mapping by conjugal mating. An important aspect of theinvention was the establishment of conditions that severely reducesuperinfection killing during selection of transductants. It is expectedthat generalized transduction will provide an important genetic tool forthe study and manipulation of this organism.

Streptomyces coelicolor phages DAH4, DAH5, and DAH6 (ATCC AccessionNumbers 203877, 203878, and 203879, respectively) and Streptomycesavermitilis phages JSN1, JSN2, and JSN3 (ATCC Accession Numbers 203874,203875, and 203876, respectively) were deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va., 20110-2209,USA, on Mar. 25, 1999. The deposits were made under the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure.

Example 1

Experimental Procedures

Bacterial strains and culture conditions. Bacterial strains used in thisstudy are listed in Table 1. Spore stocks were made from strains grownon MYM (Brawner et al., (1985) Gene, 40, 191-201). To prepare sporestocks, bacteria were streaked for isolated colonies on MYM media andincubated and 30° C. for 4 days. An isolated colony was picked andspread on MYM plates and incubated at 30° C. for 4 days or until sporeswere visible. The spores were removed with a cotton swab and stored at−20° C. Antibiotics used in the experiments described herein and theconcentrations are listed in Table 2.

TABLE 1 Bacterial Strains and Culture Conditions SPECIES STRAIN GENOTYPESOURCE Streptomyces A3(2) WT John Innes Centre coelicolor Norwich, UKStreptomyces J2402 M145, prototrophic K. Chater coelicolor SCP1⁻SCP2⁻John Innes Centre whiB::hyg Norwich, UK Streptomyces J1258 proA1 hisC9argA1 K. Chater coelicolor cysD18 uraA1 strA1 John Innes Centre Norwich,UK Streptomyces J2408 M145, prototrophic K. Chater coelicolor SCP1⁻SCP2⁻John Innes Centre whiH::ermE Norwich, UK Streptomyces YU105 proA1 argA1J. Nodwell coelicolor redE60 McMaster act::ermE whiE::hyg UniversityHamilton, Ontario Streptomyces BldK::Ω bldK::str/spc J. Nodwellcoelicolor McMaster University Hamilton, Ontario Streptomyces J222 uraA1rifA K. Chater coelicolor John Innes Centre Norwich, UK StreptomycesJ2709 proA1 hisC9 K. Chater coelicolor argA1 uraA1 John Innes CentreNorwich, UK Streptomyces J1258 proA1 hisC9 coelicolor arga1 cysD18 uraA1strA1 NF Streptomyces 1326 WT John Innes Centre lividans Norwich, UKStreptomyces TK64 proA1 John Innes Centre lividans Norwich, UKStreptomyces 10712 WT C. Stuttard venezuelae Dalhousie UniversityHalifax, NS, Canada Streptomyces JW1100 pdx C. Stuttard venezuelaeDalhousie University Halifax, NS, Canada Streptomyces JW1400 rib J.Westpheling venezuelae Athens, GA Streptomyces 32172 WT C. Denoyaavermitilis Pfizer Groton, CT Streptomyces CD1251 ermE C. Denoyaavermitilis Pfizer Groton, CT Saccharopoly- 2338 WT C. Denoya sporaerythraea Pfizer Groton, CT

TABLE 2 Antibiotics STRAIN ANTIBIOTIC CONCENTRATION J222 Rifampicin  50μg/ml J2402 Hygromycin 100 μg/ml J2408 Lincomycin 150 #g/ml Erythromycin 75 μg/ml YU105 Hygromycin 100 μg/ml Lincomycin 150 μg/ml Erythromycin 75 μg/ml CD1251 Erythromycin  5 μg/ml BldK::Ω Spectinomycin  50 μg/mlJ1258 Streptomycin  15 μg/ml

Isolation of phage. Approximately 25 grams of top soil, collected inplastic vials, was incubated with 15 mls of Actinomycete Phage Buffer(APB, 4 mM Ca(NO₃)₂, 10 mM Tris HCL, 0.005% gelatin) (Vats, S. et al.,(1987) J. Bacteriol. 169, 3809-3813) overnight at room temperature on arocking shaker. The mixture was centrifuged at 3,000 rpm for 10 minutesand the supernatant was passed through a 0.45 μm cellulose acetatefilter (Nalgene, Rochester, N.Y.). The phage-containing filtrate wasstored at 4° C. To detect phage, 100 μl of filtrate was added toStreptomyces coelicolor spores diluted to approximately 10⁷ cfu/ml. Cfurefers to colony forming unit. The mixture was added to 4 ml of NutrientSoy (Difco, Detroit, Mich.) (Nutrient Soy contains 0.3% beef extract,0.5% peptone) and 0.7% agar (NSA, also referred to as “top agar”) andpoured over Nutrient Agar (Difco, Detroit, Mich.) plates (Nutrient Agarcontains 0.3% beef extract and 0.5% peptone) and 1.5% agar, 4 mMCa(NO₃)₂, and 0.5% Dextrose (referred to as “NCG plates”). Agar wasobtained from Difco. Plates were incubated at 25° C. for 3 days andexamined for turbid plaques.

Phage were isolated by three rounds of plaque purification. From the topagar individual plaques were picked with a toothpick and streaked onto alawn of spores (10⁷ cfu) that had been spread on Nutrient Agar. Theplates were incubated at 25° C. for 3 days. This process was repeatedtwice to generate a lawn of isogenic plaque-purified phage. A starterlysate was made by adding 2 ml of APB to the lawn of plaque-purifiedphage. A sterile glass rod was used to scrape the top agar from theunderlying agar plate which was then transferred to a sterile centrifugetube, vortexed, and centrifuged at 10,000 RPM for 10 minutes to clarifylysate from cell debris. The phage-containing supernatant (lysate) wasthen transferred to a sterile tube and stored at 4° C.

Preparation of phage stocks. Phages were propagated on donor strains bystandard agar-layer techniques (Sambrook, (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, p.2.65) with APB used as phage diluent. The phage stocks were prepared byadding 100 μl of the starter lysate to 10⁷ cfu spores of the appropriatedonor strain. This mixture was then added to 4 ml of NSA and poured overNCG plates. A total of 10 plates per phage were made. The plates wereincubated at 25° C. for 5 days. The phage lawns were harvested by adding2 ml of APB to the first plate only of each phage 10 plate set, and theNSA transferred to the next plate. This process was repeated with thetop agar transferred from plate to plate in series. The phage lawns fromall 10 plates were then transferred to a centrifuge tube, vortexed, andcentrifuged as above. Phage lysates were purified by filtration througha 0.45 Nalgene cellulose acetate filter. Each phage lysate was titered(i.e., the number of phage determined) by diluting the phage in APB andspotting 20 μl of each dilution onto lawns (10⁷ cfu) of spores on NSA.The “titer” of a lysate is the number of plaque forming units (pfu) perml of lysate.

Preparation of germlings. Spores were incubated at 50° C. for 10 minutesin 0.05 M TES buffer (TES:N-tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid), pH 7.2(Hopwood, et al., (1985) Genetic Manipulation of Streptomyces—ALaboratory Manual, The John Innes Foundation, Norwich, UK, pp. 8-9). Anequal volume of 2X germination broth (GB) (2X GB: 1% yeast extract, 1%casaminoacids, and 0.01 M CaCl₂) was added, and the germlings wereincubated at 30° C. for 2 hours, centrifuged for 5 minutes at 6,000 rpmand resuspended in water. At 2 hours, a sample is removed and examinedusing a light microscope to determine if the spores are beginninggermination. The appearance of germ tubes from the spores indicatesgermination. The culture is considered germlings when about 80% of thespores show short germ tubes.

Adsorption assay. Germlings were prepared as described above. At 2hours, the germlings were centrifuged and resuspended in MYM broth, andincubated at 30° C. for an additional 4 hours. A 100 μl sample ofgermlings (about 10⁵) was taken once each hour from 0 to 6 hours. Thegermling samples were added to phage (at a concentration of 10⁵ pfu/ml)and incubated for 30 minutes at room temperature to allow foradsorption. Each mixture was then centrifuged for 5 minutes at 10,000rpm to pellet germlings and any adsorbed phages. The titer of free phageremaining in the supernatant was determined by diluting the supernatantin APB and spotting 20 μl of each dilution onto a lawn of about 10⁷spores on NSA.

UV irradiation. A kill curve was established for each phage by exposingphage suspended in APB (10⁶ pfu/ml) to ultraviolet (UV) light (250-260nm) at an intensity of 2 mW/cm²/s and sampling at 10 seconds, 20seconds, and 30 seconds. The samples containing the phage wereapproximately 6 inches from the UV light (Sylvania, Danners, Mass.).Phage were subsequently diluted in APB, and phage titers were determinedby spotting 20 μl of each dilution onto a lawn of 10⁷ S. coelicolorA3(2) spores on NSA as described herein.

Inactivation of phage with citrate. To determine phage sensitivity tosodium citrate, phage was diluted in APB and titered by spotting 20 μlof each dilution onto a lawn of 10⁷ Streptomyces coelicolor spores onNSA containing 10 mM sodium citrate. Plates were incubated at either 22°C. or 30° C. for 3 days and examined for plaques.

Genetic transduction assays. High titer (approximately 10¹⁰ pfu/ml)phage lysates were prepared on donor strains as described herein (seePreparation of phage stocks), added to recipient germlings and incubatedat room temperature for 30 minutes, then spread on supplemented minimalglucose medium (Hopwood, et al., (1985) Genetic Manipulation ofStreptomyces—A Laboratory Manual, The John Innes Foundation, Norwich,UK, p. 223) or NCG containing antibiotic and incubated at for 5-7 days22° C. Minimal glucose medium was supplemented with 0.02% trace elementssolution. Trace elements solution (100%) contains 0.004% ZnCl₂, 0.02%FeCl₃, 0.001% CuCl₂, 0.001% MgCl₂, 0.001% Na₂B₄O₇, and 0.001%(NH₄)₆Mo₄O₂₄ (Hopwood, et al., (1985) Genetic Manipulation ofStreptomyces—A Laboratory Manual, The John Innes Foundation, Norwich,UK, p. 235). Transduction frequencies were calculated as the number ofcolonies obtained after incubation for 5-7 days per pfu added to therecipient strain. To prevent superinfection killing, the phage wereeither irradiated with UV to 0.1% survival using the established killcurve described herein prior to their addition to germlings, or thephage-germlings mixture was plated on medium that contained 10 mM sodiumcitrate.

Southern hybridization analysis. Chromosomal DNA is purified fromStreptomyces using the protocol for rapid small scale isolation of totalDNA (Hopwood, et al., (1985) Genetic Manipulation of Streptomyces—ALaboratory Manual, The John Innes Foundation, Norwich, UK, pp. 72-74).Briefly, total DNA is isolated as follows. A single colony is picked andused to inoculate 50 ml of YEME broth (0.3% yeast extract, 0.5% bactopeptone, 0.3% malt extract, 1% dextrose, 34% sucrose, and 5 mM MgCl₂)which is then incubated 30° C. for 40 hours. The cells are harvested bycentrifugation at 6,000 rpm for 10 minutes. The resulting pellet is thenresuspended in 5 ml of SET buffer (75 mM NaCl, 25 mM EDTA, pH 8.0, and20 mM Tris pH 7.5). Lysozyme (1 mg/ml final concentration), is added tothe pellet suspension and incubated at 37° C. for 1 hour, at which timeProteinase K (final concentration 56 μg/ml) and sodium dodecyl sulfate(SDS, final concentration 1%,) is added to the suspension and incubatedat 55° C. for 2 hours. After incubation, NaCl (final concentration 0.8M) is added. The resulting mixture is then extracted once with an equalvolume of phenol and once with an equal volume of a 1:1 mixture ofphenol/chloroform. To the aqueous phase, Proteinase K (finalconcentration 1.5 mg/ml) and 500 mg of sarkosyl are added and theaqueous phase is incubated overnight at 37° C. The solution is thenextracted again with an equal volume of a 1:1 mixture ofphenol/chloroform, and then again with an equal volume of chloroform.Next, to precipitate the DNA, 0.1 volume of 3 M sodium acetate and 0.6volume of 2-propanol are added. The DNA can then be spooled onto asterile glass rod and suspended in about 1 ml of 10 mM Tris, pH 8.0. TheDNA is stored at 4° C.

Genomic DNA is digested with the restriction enzymes DraI and AseI(Boehringer Mannheim, Indianapolis, Ind.) following the manufacturer'sinstructions. The resulting DNA fragments are separated byelectrophoresis on a 0.8% agarose gel. The resolved DNA fragments aretransferred to a nitrocellulose membrane using technics well known tothe art (Sambrook, (1989) Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, pp. 9.31-9.55). Southernhybridizations use either a hygB gene probe or an ermE gene probe.

The hygB gene is isolated from pUH19b (obtained from Richard Seyler,University of Georgia) by cutting the plasmid with NcoI (New EnglandBioLabs, Beverly, Mass.) liberating a fragment containing the hygB gene.The fragment is gel-purified by separation by electrophoresis on 0.8%agarose gel. The hygB fragment (determined by size) is cut out of thegel, and the DNA eluted using a Promega (Madison, Wis.) Wizard DNAPurification System. An ermE gene probe is obtained and used in Southernhybridization analysis.

The probes are labeled at the 5′ end with [α-³²P] dATP using techniqueswell known to the art. Prehybridization and hybridization buffersconsist of the following (final concentrations given): 6X SSC (1X SSC is0.15 M NaCl, 0.015 M sodium citrate), 5X Denhardt's solution (50XDenhardt's: 10 grams/liter Ficoll Type 400, 10 grams/literpolyvinylpyrrolidone, 10 grams/liter bovine serum albumin Fraction V),0.1% SDS, 10 mM potassium phosphate, pH 7.2, and 250 mg/ml salmon spermDNA. Prehybridization is for 2 hours at 55° C., and hybridization isovernight at 55° C. with about 50 pmol of radiolabelled probe.Hybridization is followed by 3 consecutive washes at room temperaturefor 15 minutes each in a solution containing 2X SSC and 0.1% SDS,followed by 3 consecutive washes at 37° C. for 15 minutes each in asolution containing 1X SSC and 0.1% SDS. Kodak X-Omat scientific imagingfilm is used for autoradiography.

Phage DNA isolation and characterization. Phage DNA was prepared by themethod of Hopwood, et al., ((1985) Genetic Manipulation ofStreptomyces—A Laboratory Manual, The John Innes Foundation, Norwich,UK, pp 99-102) with the following modifications. Lysates werecentrifuged at 25,000 rpm for 90 minutes at 4° C. to sediment phage.Phage pellets were resuspended in RNAase solution (50 μg/ml in APB, theRNAse was obtained from Sigma (St. Louis, Mo.)), incubated at 37° C. for20 minutes followed by the addition of 80 μl of a 10% SDS solution andincubation at 70° C. for 30 minutes. One hundred μl of 8 M ammoniumacetate was added and the mixture was incubated for 15 minutes on ice,then centrifuged 10 minutes at 4° C. The supernatant was extracted withphenol, (1 volume supernatant:1 volume phenol), extracted with 1phenol:1 chloroform (1 volume supernatant:1 volume phenol:chloroform),and extracted with chloroform (1 volume supernatant:1 volumechloroform). The nucleic acid was precipitated with ethanol. Digestionof DNA with for instance BamHI, DraI, AseI, EcoRV, and ScpII (BoehringerMannheim, Indianapolis, Ind.) was carried out following themanufacturers instructions and separated on 0.8% agarose gel.

Results

Most wild type phage isolated from soil were found to be temperaturesensitive for lytic growth on Streptomyces coelicolor. Twenty-six soilsamples from ten different locations around Athens, Ga. were collectedand extracted with phage buffer. Samples of the extracts were tested forthe presence of plaque forming units at 30° C. Nine phages, assumed tobe different from each other because of differences in plaquemorphology, were purified. All nine phages formed turbid plaques andyielded lower titer lysates (10⁵ to 10⁷) as compared to the same phagegrown at 30° C. which formed clear, large plaques and yielded highertiter lysates (10⁸ to 10¹⁰). The turbidity of a turbid plaque was due tocells within the plaque that are not lysed by other phage in the plaque.It was distinguished from other clear plaques (plaques in which all thebacteria in the region are killed and lysed) because of the turbidcenter. A comparison of phage titers generated from Streptomycescoelicolor at 25° C. and 30° C. (Table 3) indicates that the phage arenaturally temperature sensitive for lytic growth.

TABLE 3 Temperature Sensitivity of Phage Isolated from Soil. The titerof each phage was determined at 22° C. and 30° C. in the presence andabsence of citrate. 22° C. 22° C. 30° C. 30° C. Phage −Citrate +Citrate−Citrate +Citrate DAH1 1 × 10⁵  0 2.5 × 10⁷ 500 Turbid plaques Clearplaques Turbid plaques DAH2 3 × 10⁶ 10 2.5 × 10⁷ 4 × 10⁵ Turbid plaquesTurbid Clear plaques Turbid plaques plaques DAH3 5 × 10⁶ 300  1.5 × 10⁹5 × 10⁷ Turbid plaques Turbid Clear plaques Turbid plaques plaques DAH42.5 × 10⁷   400    5 × 10⁹ 5 × 10⁶ Turbid plaques Turbid Clear plaquesTurbid plaques plaques DAH5 1 × 10⁵ 30   4 × 10⁷ 5 × 10⁷ Turbid plaquesTurbid Clear plaques Turbid plaques plaques DAH6 1 × 10⁵ 75   1 × 10⁶ 5× 10³ Turbid plaques Turbid Clear plaques Turbid plaques plaques

Phage inactivation reduces superinfection killing. The release of largenumbers of phage from infected cells during growth leads tosuperinfection. Superinfection refers to a recipient containing anucleic acid fragment from a phage particle and a nucleic acid fragmentfrom a transducing particle. Superinfection typically leads to thekilling of transductants. This is referred to as superinfection killingand the amount of superinfection killing that occurs has a dramaticeffect on the number of transductants recovered. To reducesuperinfection killing of transductants, several methods wereinvestigated to inhibit phage infection.

As shown in FIG. 1, exposure of the phage particles to UV light resultedin a sharp decrease in phage titer. All of the Streptomyces phagestested were sensitive to inactivation by UV at doses and times similarto those used for mutagenesis of phage P1. While UV light was effectiveat preventing phage infection, its potential mutagenic effects on DNAwithin transducing particles made it a less than desirable method forphage killing.

Citrate is a chelator of divalent metal ions and has been shown toprevent phage adsorption at concentrations that do not affect the growthof bacterial cells (Vats, S. et al., (1987) J. Bacteriol. 169,3809-3813). To test for sensitivity to citrate, phage were titered onNCG plates with and without sodium citrate. No plaque forming units werevisible after 1 day at 22° C. Three days after plating, small, turbidplaques (10²) were visible indicating some phage adsorption. However,the number of plaques is significantly less than on control plateswithout citrate. Dilutions of cells plated on the same medium showed noeffect on the viability of Streptomyces coelicolor.

Genetic transduction in Streptomyces coelicolor is efficient andgeneralized. Each newly isolated phage was examined for its ability tomediate transduction. Transduction assays were performed at 22° C. toreduce lytic growth of the phage. To reduce superinfection, twodifferent methods were used: 1) the phage were irradiated prior toaddition to germlings; or 2) the phage-germling mixture was plated onmedium containing 10 mM sodium citrate. Transduction of severalauxotrophic and drug resistance markers in Streptomyces coelicolor wasexamined for each phage. Surprisingly and unexpectedly, transduction ofseveral markers at frequencies ranging from 10⁻⁴ to 10⁻⁸ cfu/pfu wasdetected (Table 4). The markers transduced are positioned around theentire chromosome and the frequencies of transduction are similarsuggesting that transduction is generalized. Transduction is efficientas the transduction frequencies are at least 3 orders of magnitudehigher than the reversion frequency of the recipient strain (see Table).Also, these frequencies are well within the range of frequenciesreported for other transducing phages. For example, P22 (a wellestablished transducing phage of Salmonella) transduces markers atfrequencies ranging 10⁻⁴ to 10⁻⁸.

The ability of phage to mediate transduction was determined in otherStreptomyces spp. (Streptomyces avermitilis, Streptomyces lividans, andStreptomyces venezuelae). Surprisingly and unexpectedly, intraspecifictransduction was observed at frequencies of about 10⁻⁵ for Streptomycesavermitilis, about 10⁻⁴ to about 10⁻⁶ for Streptomyces lividans, andabout 10⁻⁶ to about 10⁻⁸ for Streptomyces venezuelae. Particularlysurprising was the observed intergeneric transduction betweenSaccharopolyspora erythraea and Streptomyces avermitilis (Table 4).

In Table 4, the germling only control is the recipient with no phageadded. This control indicates the reversion frequency of the strain,i.e., how often one would expect to see spontaneous revertants. Thephage only control is the phage with no recipient strain added. This isa test for contamination of phage lysates.

Auxotrophic markers: proA1 means that the recipient strain cannot growunless the media is supplemented with proline or a transducing phageprovided the recipient cell with the appropriate gene from the donorstrain. This is true for hisC9, argA1, and uraA1 as well; recipient willnot grow without supplemented histidine, arginine, or uracil unless thephage provided the cell with the appropriate genes from the donorstrain.

Antibiotic resistance: rifA1 means that the strain is resistant torifampicin and therefore, will grow in the presence of rifampicin.Strains without a rifA1 genotype are sensitive to rifampicin and canonly grow if a transducing phage has provided the appropriate gene fromthe donor strain. This is the same for all antibiotic markers. strAconfers resistance to streptomycin, hygB confers resistance tohygromycin and ermE confers resistance to erythromycin and lincomycin.

TABLE 4 Transduction RECIPIENT STRAIN SELECTED REVERSION TRANSDUCTIONDONOR STRAIN PHAGE RECIPIENT STRAIN MARKER FREQUENCY FREQUENCY A.Intraspecific Transduction Streptomyces coelicolor J222, uraA1 rifA1 NFDAH2 J2709,pro his arg cys ura Arginine + 3 × 10⁻⁷ J222, uraA1 rifA1 NFDAH2 Arginine + 0 J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys uraArginine + 3 × 10⁻⁶ J222, uraA1 rifA1 NF DAH4 Arginine + 0 J222, uraA1rifA1 NF DAH5 J2709,pro his arg cys ura Arginine + 3 × 10⁻⁶ J222, uraA1rifA1 NF DAH5 Arginine + 0 J222, uraA1 rifA1 NF DAH6 J2709,pro his argcys ura Arginine + 5 × 10⁻⁶ J222, uraA1 rifA1 NF DAH6 Arginine + 0J2709,pro his arg cys ura Arginine + <1 in 10⁺¹⁰ J222, uraA1 rifA1 NFDAH2 J2709,pro his arg cys ura Histidine + 8 × 10⁻⁷ J222, uraA1 rifA1 NFDAH2 Histidine + 0 J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys uraHistidine + 5 × 10⁻⁵ J222, uraA1 rifA1 NF DAH4 Histidine + 0 J222, uraA1rifA1 NF DAH5 J2709,pro his arg cys ura Histidine + 3 × 10⁻⁷ J222, uraA1rifA1 NF DAH5 Histidine + 0 J222, uraA1 rifA1 NF DAH6 J2709,pro his argcys ura Histidine + 7 × 10⁻⁶ J222, uraA1 rifA1 NF DAH6 Histidine + 0J2709,pro his arg cys ura Histidine + <1 in 10⁺¹⁰ J222, uraA1 rifA1 NFDAH2 J2709,pro his arg cys ura Proline + 3 × 10⁻⁶ J222, uraA1 rifA1 NFDAH2 Proline + J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys uraProline + 3 × 10⁻⁶ J222, uraA1 rifA1 NF DAH4 Proline + 0 J222, uraA1rifA1 NF DAH5 J2709,pro his arg cys ura Proline + 7 × 10⁻⁶ J222, uraA1rifA1 NF DAH5 Proline + 0 J222, uraA1 rifA1 NF DAH6 J2709,pro his argcys ura Proline + 5 × 10⁻⁶ J222, uraA1 rifA1 NF DAH6 Proline + 0J2709,pro his arg cys ura Proline + <1 in 10⁺¹⁰ J222, uraA1 rifA1 NFDAH2 A3(2), WT Rifampicin 5 × 10⁻⁶ resistance J222, uraA1 rifA1 NF DAH2Rifampicin 0 resistance J222, uraA1 rifA1 NF DAR4 A3(2), WT Rifampicin 3× 10⁻⁶ resistance J222, uraA1 rifA1 NF DAR4 Rifampicin 0 resistanceJ222, uraA1 rifA1 NF DAH5 A3(2), WT Rifampicin 7 × 10⁻⁶ resistance J222,uraA1 rifA1 NF DAH5 Rifampicin 0 resistance J222, uraA1 rifA1 NF DAH6A3(2), WT Rifampicin 5 × 10⁻⁶ resistance J222, uraA1 rifA1 NF DAH6Rifampicin 0 resistance A3(2), WT Rifampicin <1 in 10⁺¹⁰ resistanceStreptomyces avermitilis CD1251 JSN 31272, WT Lincomycin 4 × 10⁻⁵resistance CD1251 JSN Lincomycin 0 resistance CD1251 JSN3 31272, WTLincomycin 3 × 10⁻⁵ resistance CD1251 JSN3 Lincomycin 0 resistance31272, WT Lincomycin <1 in 10⁺⁹ resistance Streptomyces venezuelae10712, WT SV1 JW1400, rib Riboflavin + 0 10712, WT SV1 Riboflavin + 010712, WT BTH JW1400, rib Riboflavin + 0 10712, WT BTH Riboflavin + 010712, WT MRT JW1400, rib Riboflavin + 7 × 10⁻⁸ 10712, WT MRTRiboflavin + 0 JW1400, rib Riboflavin + <1 in 10⁺¹⁰ 10712, WT SV1JW1100, pdx Pyridoxal + 0 10712, WT SV1 Pyridoxal + 0 10712, WT BTHJW1100, pdx Pyridoxal + 2.5 × 10⁻⁸ 10712, WT BTH Pyridoxal + 0 10712, WTMRT JW1100, pdx Pyridoxal+ 2× 10⁻⁶ 10712, WT MRT Pyridoxal + 0 JW1100,pdx Pyridoxal+ <1 in 10⁺¹⁰ Streptomyces lividans 1326, WT DAH2 TK64Proline + 3 × 10⁻⁶ 1326, WT DAH2 Proline + 0 1326, WT DAH3 TK64Proline + 2 × 10⁻⁶ 1326, WT DAH3 Proline + 0 1326, WT DAH4 TK64Proline + 4 × 10⁻⁵ 1326, WT DAH4 Proline + 0 1326, WT DAH5 TK64Proline + 1 × 10⁻⁶ 1326, WT DAHS Proline + 0 1326, WT DAH6 TK64Proline + 1 × 10⁻⁶ 1326, WT DAH6 Proline + 0 TK64 Proline + <1 in 10⁺¹⁰B. Interspecific Transduction Saccharopolyspora JSN1 31272, WTLincomycin 4× 10⁻⁴ erythraea, WT resistance Saccharopolyspora JSN1Lincomycin 0 erythraea, WT resistance Saccharopolyspora JSN2 31272, WTLincomycin 3 × 10⁻⁶ erythraea, WT resistance Saccharopolyspora JSN2Lincomycin 0 erythraea, WT resistance Saccharopolyspora JSN3 31272, WTLincomycin 4× 10⁻⁴ erythraea, WT resistance Saccharopolyspora JSN3Lincomycin 0 erythraea, WT resistance 31272, WT Lincomycin >1 in 10⁺⁹resistance

Co-transduction confirms linkage established by conjugal mating forseveral genetic markers. To examine co-transduction (i.e., the transferof two genetic markers at the same time) of markers by these phage,genes identified by mutations that had been previously mapped usingconjugal mating or physical mapping were used in combination with eachother and with drug resistance markers that had been introduced intoknown locations within the chromosome. As shown in Table 5,co-transduction was observed at frequencies that are in good agreementwith previously reported linkage.

TABLE 5 Cotransduction RECIPIENT STRAIN RECIPIENT SELECTED REVERSIONTRANSDUCTION SCREENED COTRANSDUCTION DONOR STRAIN PHAGE STRAIN MARKERFREQUENCY FREQUENCY MARKER FREQUENCY YU105, pro arg red DAH5 bldK::ΩLincomycin 2 × 10⁻⁸ Spectinomycin 100% act::ermE whiE::hyg resistancesensitivity YU105, pro arg red DAH5 Lincomycin 0 act::ermE whiE::hygresistance YU105, pro arg red DAH6 bldK:Ω Lincomycin 3 × 10⁻⁸Spectinomycin 100% act::ermE whiE::hyg resistance sensitivity YU105, proarg red DAH6 Lincomycin 0 act::ermE whiE::hyg resistance bldK::ΩLincomycin <1 in 10⁺⁹ resistance argA1 cysD18 uraA1 DAH5 J222Streptomycin 2 × 10⁻⁶ Rifampicin  50% straA1 NF resistance sensitivityargA1 cysD18 uraA1 DAH5 Streptomycin 0 straA1 NF resistance argA1 cysD18uraA1 DAH6 J222 Streptomycin 1 × 10⁻⁵ Rifampicin 100% straA1 NFresistance sensitivity argA1 cysD18 uraA1 DAH6 Streptomycin 0 straA1 NFresistance J222 Streptomycin <1 in 10⁺⁹ resistance

Physical analysis of transductants confirms DNA transfer. To examine thephysical location of transduced markers in the recipient chromosome,Southern hybridization experiments are performed. DNA is isolated fromS. coelicolor strains J2402, which contains a hygB insertion into thewhiB gene, and J2408, which contains an ermE insertion into the whiHgene. Phage grown on strain J2402 is added to J2408 germlings, and cellsthat are resistant to both hygromycin (encoded by hygB) and lincomycin(encoded by ermE) are isolated from cultures at a frequency above thereversion frequency. No doubly resistant cells are recovered fromcontrol cultures (no phage added) in any experiment, which stronglysuggests that the phage provide the recipient cells with the gene thatconfers resistance to hygromycin from the donor strain. To confirm thatthe transductants contained the transferred drug resistance gene, DNAsisolated from randomly selected transductants are analyzed by Southernblotting using the hygB drug resistance gene probe. DNA hybridizationpatterns of the transductants is the same as those of the donor strain,while no hybridization is seen with the recipient strain. A gene probefor the ermE gene present in the recipient strain hybridizes with theDNA from the transductants, but does not hybridize with the donorstrain. These experiments demonstrate that chromosomal DNA from thedonor strain is transferred by the phage to the transductant strain andintegrated in the host chromosome.

Transduction was not detected when assays were performed at 30° C. Thesephages were exceptional in their ability to grow lytically at 30° C.,the growth temperature typically used to incubate S. coelicolor and infact most streptomycete strains. If lawns with only a few plaques wereincubated at 30° C., the entire lawn was lysed in a few days. Extensivesuperinfection killing resulting from this active lytic growth mightexplain the failure to detect transduction in many Streptomyces species.In fact, when transduction assays were performed exactly as describedabove but at 30° C., no transductants were detected. It is very likelythat superinfection killing does, in fact, reduce or eliminate theability of transductants to survive.

Preliminary physical characterization of the transducing phages revealsthat they are different from each other. To determine the size of thephage genomes, nucleic acid was extracted from four phage, DAH2, DAH4,DAH5, and DAH6, using a standard approach for Streptomyces phages(Hopwood, et al., (1985) Genetic Manipulation of Streptomyces—ALaboratory Manual, The John Innes Foundation, Norwich, UK, pp. 99-102).In all cases the nucleic acid isolated from the phages was digested withseveral restriction enzymes thus indicating that it is double strandedDNA. The genome sizes were estimated using digestion with restrictionendonucleases and separation of fragments by agarose gelelectrophoresis. The size of DNA isolated from DAH2, DAH4, DAH5, andDAH6 was about 60 kilobases (kb), 45 kb, 45 kb, and 25 kb, respectively.The differences in size strongly suggest that there are at least threedifferent types of phage. Moreover, the differences in transductionfrequencies and the differences in plaque morphology between DAH4 andDAH5 strongly suggest that these two phages are not the same phage,despite the similar size of the DNA. Thus, DAH2, DAH4, DAH5, and DAH6are each unique.

The complete disclosures of all patents, patent applications,publications, and nucleic acid and protein database entries, includingfor example GenBank accession numbers and EMBL accession numbers, thatare cited herein are hereby incorporated by reference as if individuallyincorporated. Various modifications and alterations of this inventionwill become apparent to those skilled in the art without departing fromthe scope and spirit of this invention, and it should be understood thatthis invention is not to be unduly limited to the illustrativeembodiments set forth herein.

What is claimed is:
 1. An isolated generalized transducing phage thatcan transfer at least one nucleic acid sent from a donor Actinomycetalesmicrobe to a recipient Actinomycetales microbe at a temperature of lessthan 28° C., wherein the frequency of transduction is at least 10⁻⁵. 2.The phage of claim 1 wherein either the donor or recipientActinomycetales is a Streptomycetaceae.
 3. The phage of claim 2 whereinthe Streptomycetaceae is a Streptomyces.
 4. The phage of claim 1 whereinthe donor microbe and the recipient microbe are members of differentgenera.
 5. The phage of claim 1 wherein the donor microbe and therecipient microbe are members of different species.
 6. The phage ofclaim 1 wherein the donor microbe and the recipient microbe are membersof the same species.
 7. A method for screening for a transducing phage,the method comprising: combining a phage with a donor Actinomycetalesmicrobe forming a first mixture and incubating the first mixture underconditions such that a lystate is produced; combining the lysate with arecipient Actinomycetales microbe forming a second mixture andincubating the second mixture at less than 28° C., wherein the donormicrobe contains a coding region encoding a marker and the recipientmicrobe does not contain the coding region; and detecting a recipientmicrobe containing the coding region, wherein the detection of therecipient microbe containing the coding region indicates the phage is atransducing phage.
 8. The method of claim 7 wherein the donorActinomycetales or the recipient Actinomycetales is a Streptomycetaceae.9. The method of claim 8 wherein the Streptomycetaceae is aStreptomyces.
 10. The method of claim 7 wherein the donorActinomycetales and the recipient Actinomycetales are selected from thegroup consisting of spores, mycelial fragments, germlings, protoplast,and mixtures thereof.
 11. The method of claim 7 wherein detecting therecipient microbe containing the coding region comprises detecting acolony on a plate.
 12. The method of claim 7 further comprising reducingsuperinfection of the recipient microbe containing the coding region.13. The method of claim 12 wherein reducing superinfection comprisesreducing superinfection killing of the recipient microbe containing thecoding region.
 14. The method of claim 13 wherein reducingsuperinfection killing of the recipient microbe containing the codingregion comprises treating the lysate prior to combining it with therecipient microbe.
 15. The method of claim 14 wherein treating thelysate comprises exposing it to ultraviolet radiation.
 16. The method ofclaim 13 wherein reducing superinfection killing of the recipientmicrobe containing the coding region comprises treating the recipientmicrobe containing the coding region with a chelator.
 17. The method ofclaim 16 wherein the chelator comprises citrate.
 18. The method of claim7 wherein the transducing phage is a generalized transducing phage. 19.The method of claim 7 wherein the transducing phage is a specializedtransducing phage.
 20. The method of claim 7 wherein the phage isobtained from the group consisting of soil, water, organic material,decomposing organic material, and volcanic ash.
 21. The method of claim20 wherein the phage is plaque purified prior to forming the firstmixture.
 22. A method for screening for a transducing phage, the methodcomprising: combining an isolated phage DNA with a donor Actinomycetalesmicrobe competent to take up DNA forming a first mixture and incubatingthe first mixture under conditions such that the donor Actinomycetalesmicrobe takes up the isolated phage DNA; incubating the mixture underconditions such that a lystate is produced; combining the lysate with arecipient Actinomycetales microbe forming a second mixture andincubating the second mixture at less than 28° C., wherein the donormicrobe contains a coding region encoding a marker and the recipientmicrobe does not contain the coding region; and detecting a recipientmicrobe containing the coding region, wherein the detection of arecipient microbe containing the coding region indicates the phage is atransducing phage.
 23. An isolated phage deposited under ATCC AccessionNumber
 203877. 24. An isolated phage deposited under ATCC AccessionNumber
 203878. 25. An isolated phage deposited under ATCC AccessionNumber
 203879. 26. An isolated phage deposited under ATCC AccessionNumber
 203874. 27. An isolated phage deposited under ATCC AccessionNumber
 203875. 28. An isolated phage deposited under ATCC AccessionNumber 203876.